Article

pubs.acs.org/Biomac

Well-Defined Cholesterol Polymers with pH-Controlled Membrane

Switching Activity
Sema Sevimli,†,‡,& Fatih Inci,†,§ Hadi M. Zareie,∥,⊥ and Volga Bulmus*,†,#,○
†
School of Biotechnology and Biomolecular Sciences, ‡The Centre for Advanced Macromolecular Design, and &Australian Centre for
Nanomedicine (ACN), The University of New South Wales, Sydney, NSW 2052, Australia
§
Department of Molecular Biology-Genetics and Biotechnology Program (MOBGAM), Istanbul Technical University, Istanbul
34469, Turkey
∥
Department of Electrical-Electronics Engineering and Nanotechnology Graduate Program, Gediz University, Izmir 35665, Turkey
⊥
Microstructural Analysis Unit, School of Physics and Advanced Materials, University of Technology, Sydney, Altimo NSW 2007,
Australia
#
Department of Chemical Engineering and ○Biotechnology and Bioengineering Graduate Program, Izmir Institute of Technology,
Urla, Izmir 35430, Turkey
*
S Supporting Information

ABSTRACT: Cholesterol has been used as an effective

component of therapeutic delivery systems because of its
ability to cross cellular membranes. Considering this, well-
defined copolymers of methacrylic acid and cholesteryl
methacrylate, poly(methacrylic acid-co-cholesteryl methacry-
late) P(MAA-co-CMA), were generated as potential delivery
system components for pH-controlled intracellular delivery of therapeutics. Statistical copolymers with varying cholesterol
contents (2, 4, and 8 mol %) were synthesized via reversible addition−fragmentation chain transfer (RAFT) polymerization.
Dynamic light scattering (DLS) analysis showed that the hydrodynamic diameters of the copolymers in aqueous solutions ranged
from 5 ± 0.3 to 7 ± 0.4 nm for the copolymers having 2 and 4 mol % CMA and 8 ± 1.1 to 13 ± 1.9 nm for the copolymer having
8 mol % CMA with increasing pH (pH 4.5−7.4). Atomic force microscopy (AFM) analysis revealed that the copolymer having 8
mol % CMA formed supramolecular assemblies while the copolymers having 2 and 4 mol % CMA existed as unimers in aqueous
solution. The pH-responsive behavior of the copolymers was investigated via UV−visible spectroscopy revealing phase
transitions at pH 3.9 for 2 mol % CMA, pH 4.7 for 4 mol % CMA, and pH 5.4 for 8 mol % CMA. Lipid bilayers and liposomes as
models for cellular membranes were generated to probe their interactions with the synthesized copolymers. The interactions
were determined in a pH-dependent manner (at pH 5.0 and 7.4) using surface plasmon resonance (SPR) spectroscopy and
liposome leakage assay. Both the SPR analyses and liposome leakage assays indicated that the copolymer containing 2 mol %
CMA displayed the greatest polymer−lipid interactions at pH 5.0, presenting the highest binding ability to the lipid bilayer
surfaces, and also demonstrating the highest membrane destabilization activity. CellTiter−Blue assay showed that the copolymers
did not affect the cell viability up to 30 μM over a period of 72 h.

■ INTRODUCTION
A number of potent therapeutic strategies rely on effective
transport of macromolecular therapeutics, such as proteins/
peptides and DNA/RNA, into cells. However, most macro-
molecular therapeutics cannot diffuse passively through cellular
membranes because of their large hydrodynamic size and
hydrophilic nature.1−6 Various systems such as lipids,7−9
endosome-fusogenic peptides,10−13 and endosome-lytic poly-
mers14−26 have been developed to increase the intracellular
transport of macromolecular therapeutics.
pH-responsive membrane-destabilizing polymers have im-
proved the intracellular delivery of therapeutics both in
vitro27−29 and in vivo.30 These polymers that mimic the pH-
controllable membrane activity of endosome-fusogenic peptides
combine hydrophobic and acidic units in their structure and
display a transition from a hydrophilic state to a lipophilic state
at acidic pHs, enhancing interactions with cell membrane.
Poly(methacrylic acid), a pH-responsive polymer, has been
widely utilized as an essential constituent for generating pH-
sensitive therapeutic delivery systems.31−34
When compared with peptides and synthetic polymers, lipid-
based systems have been more effective in interacting with
cellular membranes and enhancing the cytoplasmic delivery of
therapeutics. The naturally occurring hydrophobic building
blocks35 are essential for enhancing stability,36 stimulating self-
assembly,37,38 increasing cellular uptake via endocytosis
mechanisms,39 disrupting endosomal membranes along with
facilitating effective transfection of macromolecular therapeu-
Received: May 31, 2012
Revised: August 23, 2012
Published: August 24, 2012

© 2012 American Chemical Society 3064 dx.doi.org/10.1021/bm300846e | Biomacromolecules 2012, 13, 3064−3075
Biomacromolecules
Table 1. Experimental Conditions for Copolymerization of t-BMA and CMAa
total monomer concentration (M) [t-BMA]/[CMA]/[RAFT]/[AIBN] (mole ratio)
1.25 40.0/10.0/1.0/0.2
4.08 200.0/4.0/1.0/0.2
4.08 200.0/4.0/1.0/0.2
8.20 200.0/5.0/1.0/0.2
8.20 200.0/5.0/1.0/0.2
a
Polymerization conditions: temperature = 68 °C, solvent = toluene. bMonomer conversion determined by 1H NMR. cThe number average
molecular weight determined by THF GPC analysis is using PS standards. dPolydispersity index. eThe theoretical molecular weight calculated by
Mn,theo = ([M]o/[RAFT]o) × conversion × MWmonomer, where [M]o,[RAFT]o, conversion, MWmonomer are the initial monomer and RAFT agent
concentrations, monomer conversion, and molecular weight of the monomer, respectively.
tics,8,4041 and generating carriers which are well tolerated in
biological systems.42 For example, cholesterol, which accounts
for 20−25% of the lipid molecules in the cell membrane,43
interacts easily with the cell membrane. It is easily transported
into the cells by lipoproteins and albumin, usually via
endocytosis mechanism. In a number of studies,8,41 cholesterol
conjugation has proved to facilitate the entry of macro-
molecular therapeutics into the cells.
Considering the ability of cholesterol to cross cellular
membranes, we prepared well-defined copolymers of meth-
acrylic acid and cholesteryl methacrylate, poly(methacrylic acid-
co-cholesteryl methacrylate) P(MAA-co-CMA), as potential
components of delivery systems for pH-controlled delivery of
therapeutics into cells. Hence, the ability of cholesterol to
interact with lipid membranes was potentially synergized with
multiplicity and pH-sensitivity in a copolymer structure. The
reversible addition−fragmentation chain transfer (RAFT)
polymerization44−48 was used to synthesize well-defined
copolymers with controlled molecular weight and narrow
molecular weight distribution. Aqueous solution properties of
the copolymers were investigated by varying techniques
revealing information on hydrodynamic size, conformation,
and phase transition as a function of pH. The pH-dependent
membrane-destabilizing activities of copolymers were examined
by the liposome leakage assay and further lipid layer
interactions were explored by surface plasmon resonance
(SPR) spectroscopy. Furthermore, the cytotoxicity of copoly-
mers with varying cholesterol content was investigated using a
human neuroblastoma cell line. Overall, the results support the
value of these new copolymers for further investigations as
components of delivery systems for transporting therapeutics
through cellular membranes.
■
EXPERIMENTAL SECTION
Materials. The initiator, 2,2-azobisisobutyronitrile (AIBN), was
recrystallized twice from methanol prior to use. High purity nitrogen
(Linde gases, 99.99%) was used for removing oxygen from the
polymerization solutions. tert-Butyl methacrylate (t-BMA) monomer
(Aldrich, 99%) was purified via basic alumina gel column
chromatography before use. Triethylamine (Sigma-Aldrich, 99%)
was stored with sodium hydroxide pellets (Univar) for 2 days prior
to use. Cholesterol (Sigma, 98%), methacryloyl chloride (Fluka,
>97%), dichloromethane (Univar, analytical grade reagent), toluene
(Univar, analytical grade reagent), methanol (Univar, analytical grade
reagent), acetone (Univar, analytical grade), trifluoro acetic acid
(Sigma), Triton X100 (Sigma), phosphate buffer saline (PBS) pellets
(Sigma), potassium dihydrogen phosphate (Univar), sulforhodamine
B (Sigma), egg yolk phosphatidylcholine (Sigma), chloroform
(Sigma), hexadecanethiol (Sigma), Dulbecco’s modified Eagle medium
(DMEM; Lonza), 10% Australian fetal bovine serum (FBS; Lonza),
and CellTiter-Blue cell viability assay (Promega) were used as
received. Cholesteryl methacrylate (CMA) monomer was synthesized
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time (h) conversionb (%) Mnc(g/mol) PDId Mn,theoe
20 74 4000 1.13 4200
6 48 9600 1.10 14500
9 60 11900 1.14 18150
6 56 10500 1.14 16900
9 64 14000 1.12 19200
according to a modified procedure described previously in the
literature (Figure S1, Supporting Information).49,50 The RAFT agent,
4-(cyanopentanoic acid)-4-dithiobenzoate (CPADB), was synthesized
according to the procedure reported in the literature.51,52 Membranes
for dialysis (MWCO 3500) were purchased from Fisher Biotech
(Cellu SepT4, regenerated cellulose-Tubular membrane).
Analytical Techniques. Nuclear Magnetic Resonance (NMR)
Spectroscopy. All NMR spectra were taken using Bruker 300 MHz
NMR spectrometer. According to their solubility, samples were
analyzed in CDCl3, DMSO-d6, or D2O as the NMR solvents.
Gel Permeation Chromatography (GPC). Gel permeation
chromatography was performed using HPLC grade tetrahydrofuran
(THF) as the mobile phase. Polymer solutions (3−5 mg/mL in THF)
were injected to GPC at 40 °C (flow rate = 1 mL/min). A Shimadzu
modular system comprising of a SIL-10AD autoinjector, a PL 5.0 mm
bead-size guard column (50 × 7.8 mm) followed by four linear PL
(Styragel) columns (105, 104, 103, and 500 Å) and a RID-10A
differential refractive-index detector was used. Calibration was
performed with commercial polystyrene standards ranging from 500
to 106 g/mol.
UV−Visible (UV−vis) Spectrophotometer. UV−visible spectra were
obtained by a double beam Hitachi−UV spectrometer (Model No.: U-
2800) using UV solutions 2.1 software. The absorbance of polymer
solutions (0.125 mM) in buffer solutions with varying pH values was
measured at 400 nm using quartz cuvettes.
Differential Scanning Calorimeter (DSC). DSC analysis was
performed using a Perkin-Elmer Pyris 1 under N2 atmosphere, from
−10 to 250 °C at a heating rate of 15 °C/min. High purity indium was
used to calibrate the calorimeter. Thermal history difference was
erased by reheating the sample and recording a second DSC scan; 10−
15 mg polymer was used for each running.
Dynamic Light Scattering (DLS). Dynamic light scattering studies
were performed using a Malvern Zetasizer NaNo ZS Instrument
(Malvern, U.S.A.) equipped with a 4 mV He−Ne laser operating at λ =
633 nm, an avalanche photodiode detector with high quantum
efficiency, and an ALV/LSE-5003 multiple tau digital correlator
electronics system. The polymer sample solutions were prepared at
0.125 mM concentration.
Atomic Force Microscopy (AFM). Atomic force microscopy (AFM)
studies were performed using a Nanomagnetic AFM apparatus in
tapping mode. A total of 20 μL of the copolymer solution (0.125 mM)
at pH 5.0 was placed on silicon substrate, and the sample was dried
under atmospheric conditions at room temperature. The scans were
typically done at rates between 1 and 4 Hz. The images were obtained
using a silicon nitride cantilever with a nominal force constant of 0.38
N/m.
fMax Fluorescence Spectroscopy. A fMax Fluorescence Plate
Reader from fMax Fluorescence Microplate Molecular Devices
Corporation was used for measurements in Liposome Lysis Assay
and CellTiter-Blue Assay with fluorescence readings measured at
488λex/585λem nm and 540λex/590λem nm, respectively. Measurements
were performed by using the fMax Fluorescence user software
program.
Methods. The procedure for the RAFT homopolymerizations of
cholesteryl methacrylate (CMA) and tert-butyl methacrylate (t-BMA)
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was described in Supporting Information (Schemes S1 and S2 and
Figures S2 and S3).
RAFT Copolymerization of tert-Butyl Methacrylate (t-BMA) and
Cholesteryl Methacrylate (CMA). The copolymerizations were
conducted at varying comonomer concentrations as shown in Table
1. t-BMA and CMA monomers, initiator (2,2-azobisisobutyronitrile
(AIBN)), and RAFT agent (4-(cyanopentanoic acid)-4-dithiobenzoate
(CPADB)) were dissolved in toluene. The solution was sealed with a
rubber septum and then degassed using nitrogen for 30 min in an ice
bath. The polymerization was carried out by immersing the vial into a
preheated oil bath of 68 °C. At predetermined time intervals
polymerization solution was drawn out from the vial using a syringe
for further analysis. The monomer conversion was determined by 1H
NMR analysis of the crude polymerization mixtures. The polymer
sample was purified by precipitation twice in methanol and dried
under vacuo overnight. 1H NMR spectrum of the purified product is
shown in Supporting Information (Figure S7A).1H NMR (CDCl3, 300
MHz) ppm: 5.3 (d, 1H, -CCH-, olefin group in cholesterol), 4.5 (m,
1H, -COO-CH-), 2.3 (d, 2H, -CH-CH2-), 1.81 (s, 2H, -C-CH2-), 1.41
(s, 9H, -C-(CH3)3), 1.02 (s, 3H, -C-CH3), 0.93 (d, 3H, -CH-CH3),
0.88−0.84 (q, 6H, -CH-(CH3)2), 0.68 (s, 3H, -C-CH3). The molecular
weight of the purified polymers was obtained by gel permeation
chromatography (GPC) using THF as the mobile phase.
Hydrolysis of Poly(tert-butyl methacrylate-co-cholesteryl meth-
acrylate) to Poly(methacrylic acid-co-cholesteryl methacrylate).
The copolymer P(t-BMA-co-CMA) (325 mg, 2.3 mmol tert-butyl
ester) was dissolved in dichloromethane (DCM; 5 mL). The mixture
was allowed to stir for 10 min to dissolve the polymer. Trifluoro acetic
acid (TFA; 0.894 mL, 114 mmol) was slowly added while vigorously
stirring. The reaction continued to stir for 32 h at room temperature.
Excess TFA along with DCM was removed at room temperature with
air flowing through the vial and dried under vacuum overnight.
Removal of tert-butyl group from the copolymer was verified by 1H
NMR
1
H NMR (DMSO, 300 MHz) ppm: 12.3 (s, 1H, -CO-OH), 5.3 (d,
1H, -CCH-, olefin group in cholesterol), 4.5(m, 1H, -COO-CH-),
2.3 (d, 2H, -CH-CH2-), 1.82 (s, 2H, -C-CH2-), 1.02 (s, 3H, -C-CH3),
0.93 (d, 3H, -CH-CH3), 0.88−0.84 (q, 6H, -CH-(CH3)2), 0.68 (s, 3H,
-C-CH3).
To obtain the final product, the copolymer, poly(methacrylic acid-
co-cholesteryl methacrylate) P(MAA-co-CMA), was dissolved in basic
aqueous solution (10 mM NaOH solution prepared with deionized
water) and transferred to dialysis tubing (MWCO: 3500). The
solution was dialyzed against deionized water for 3 days followed by
freeze-drying giving a white powder. The average yield was 90% for
overall hydrolysis process. The final polymer was analyzed by 1H
NMR (Figure S7B, Supporting Information).
1
H NMR (D2O, 300 MHz) ppm: 5.3 (d, 1H, -CCH-, olefin
group in cholesterol), 4.5 (m, 1H, -COO-CH-), 1.81 (s, 2H, -C-CH2-),
1.02 (s, 3H, -C-CH3), 0.68 (s, 3H, -C-CH3).
Turbidity Assay. The pH-responsive phase behaviors of polymers
were studied by measuring the turbidity change of polymer solutions
at varying pH values via UV−visible spectroscopy.53 For spectropho-
tometric analysis, citrate buffer solutions in the pH range of 3.0−7.0
were prepared by mixing citric acid (0.1 M) and dibasic sodium
phosphate (0.2 M) aqueous solutions. A phosphate buffer solution
(0.1 M) at pH 7.4 was prepared by mixing sodium phosphate
monobasic (0.1 M) and sodium phosphate dibasic (0.1 M) aqueous
solutions. The ionic strength of the buffer solutions were adjusted to
0.1 M by the addition of NaCl to yield isotonic solutions. Three
different P(MAA-co-CMA) samples with varying cholesterol content,
(2 mol % CMA, with number average molecular weight (Mn)GPC of
16500 g/mol and polydispersity index (PDI) of 1.19; 4 mol % CMA,
with (Mn)GPC 15800 g/mol and PDI 1.10; 8 mol % CMA, with
(Mn)GPC 18000 g/mol and PDI 1.11) were dissolved in buffer
solutions. The final copolymer concentration was 0.125 mM. The
absorbance of each polymer solution from acidic pH to neutral pH was
detected by a UV−visible spectrophotometer at 400 nm.
Surface Plasmon Resonance. Surface plasmon resonance (SPR)
experiments were performed using a Biacore 2000 system (GE
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Healthcare Biacore) to monitor polymer binding onto lipid bilayers in
real-time. SPR gold slides, used for lipid bilayer immobilization, were
first washed with ethanol, treated with 0.1 mM hexadecanethiol/DCM
solution overnight (to produce self-assembled monolayer of
hexadecanethiol), then rinsed thoroughly with DCM, and finally
dried under a gentle stream of nitrogen.
Liposomes of different compositions were prepared to mimic both
the cell plasma membrane (composed of cholesterol/phosphatidylcho-
line constituents at a cholesterol/phosphatidylcholine molar ratio of
0.42) and endosome membrane (molar ratio of 0.80) lipid
arrangements.54 Small, unilamellar vesicles (SUV) were prepared by
sonication, according to the protocol described by Morrissey
Laboratories.55 In brief, egg yolk phosphatidylcholine (8.4 mg, 0.01
mmol for plasma membrane; 7 mg, 0.08 mmol for endosome
membrane) and cholesterol (1.6 mg, 0.04 mmol for plasma
membrane; 3 mg, 0.07 mmol for endosome) were solubilized in 1
mL of chloroform (in a round-bottom flask) and dried under nitrogen,
generating a lipid film that was stored overnight at +4 °C. The thin
lipid film was resuspended in 5 mL phosphate buffered (pH 7.4) or
citrate-phosphate buffer (pH 5.0) by vortex mixing, hydrated for 1 h at
room temperature and then sonicated forming SUVs.
Liposomes were injected to the SPR at 20 μL/min for 6.5 min until
the surfaces were saturated. The solution was allowed to sit on the
surface for 20 min. During this period, liposomes were attached to self-
assembled hexadecane monolayer surface and then disrupted to form
lipid bilayer on the surface. Surfaces covered with lipid bilayer were
then washed with several pulses (ranging from 30 s to 5 min) of
related buffer (citrate-phosphate buffer at pH 5.0 or phosphate buffer
at pH 7.4) to remove loosely bound lipids and stabilize the surfaces.
Polymer solutions (2 mol % CMA, with (Mn)GPC of 16500 g/mol; 4
mol % CMA with (Mn)GPC 15800 g/mol; 8 mol % CMA with (Mn)GPC
18000 g/mol) prepared at 0.5 mM concentration in related buffer
solutions (citrate-phosphate buffer at pH 5.0 or phosphate buffer at
pH 7.4) were injected over the surfaces at 20 μL/min flow rate,
allowing real-time monitoring of the interactions between polymers
and lipid bilayers of different composition in acidic and neutral
medium. Lipid bilayer surfaces that reached saturation point were
rinsed with 6.5 min injections of their related buffer solutions.
Liposome Leakage Assay. Liposomes composed of cholesterol/
phosphatidylcholine constituents at a cholesterol/phosphatidylcholine
molar ratio of 0.42 were prepared intending to mimic the cell plasma
membrane lipid composition.54 The liposomes were prepared by
sonication according to a protocol described by Morrissey
Laboratories.55 Briefly, egg yolk phophatidylcholine (42 mg, 0.05
mmol) and cholesterol (8 mg, 0.02 mmol) were dissolved in 1 mL
chloroform and placed in a round-bottom flask. A thin lipid film was
generated on the bottom of the flask by evaporation of the chlorofom
via nitrogen gas and stored overnight at +4 °C. The lipid film was
hydrated with sulforhodamine B (SRB; 2.5 mM, 2.5 mL) solution in
phosphate buffer saline (pH 7.4) and then vortexed vigorously. The
lipid solution was incubated for 1 h at room temperature followed by
sonication generating small, unilamellar vesicles (SUV) with diameters
in the range of 15−50 nm. Untrapped SRB molecules were eliminated
by size exclusion chromatography on a Sephadex G-25 column (bed
volume 8.3 mL, bed height 5 cm; PD-10) using phosphate buffer saline
as the eluting solution. 750 μL of the SRB loaded liposomes were
added to the isotonic buffer solution (3.75 mL, citrate-phosphate
buffer at pH 5.0 or phosphate buffer at pH 7.4). The solution was
equally divided into 6 vials (750 μL) and the final volume was made
up to 900 μL by addition of isotonic buffer solution (in the absence of
polymer), Triton X-100 solution (10% in Milli-Q water) or copolymer
solution prepared in Milli-Q water at 1.5 μM or 3.0 μM concentration
(2 mol % CMA, with (Mn)GPC of 16500 g/mol; 4 mol % CMA with
(Mn)GPC 15800 g/mol; 8 mol % CMA with (Mn)GPC 18000 g/mol or
poly(methacrylic acid), PMAA, cholesterol mole content 0% with
(Mn)GPC 36000 g/mol). The reason for using dilute copolymer
solutions were to determine the effectiveness of the copolymers in
selective (pH-dependent) membrane destabilization, as detailed in
Results and Discussion. After 1 h incubation, the solutions were
transferred to a 96-well plate and the fluorescent intensity (λex = 488
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Biomacromolecules
Scheme 1. Synthesis of P(t-BMA-co-CMA) via RAFT Polymerization and Subsequent Acid Hydrolysis, Yielding
Poly(methacrylic acid-co-cholesteryl methacrylate) (P(MAA-co-CMA))
nm, λem = 585 nm) of the resulting solutions was measured by a fMax
fluorescence plate reader (fMax Fluorescence Microplate Molecular
Devices Corporation). In a control experiment, a series of SRB-loaded
liposomes were analyzed by the same protocol without using polymer.
This experiment suggested that SPR-loaded liposomes, without
polymer treatment, were stable in isotonic buffer solutions (pH 5.0
and 7.4) as they showed similar fluorescence trends in both
environments. The calibration curves of SRB fluorescence were
obtained at both pH values prior to the liposome leakage experiment.
Percent leakage was defined as
%leakage = (Fp − F0)/(F100 − F0) × 100
where F0 and Fp are the fluorescence intensity of blank solution (SRB-
loaded liposomes in buffer solutions without polymer) and polymer
solutions (SRB-loaded liposomes in buffer solutions with polymer),
respectively. A 100% leakage, F100, was taken as the fluorescence
intensity of the SRB-loaded liposome solutions after the addition of
10% Triton X-100, which corresponded to a complete liposome lysis.
Determination of Cell Viability via CellTiter-Blue Assay. Human
neuroblastoma (SH-EP) cell line was used to determine the toxicity of
the polymers. The cells were grown in Dulbecco’s modified Eagle
medium (DMEM) containing 4.5 g/L glucose and L-glutamine,
supplemented with 10% Australian Fetal Bovine Serum (FBS). Cells
were grown at 37 °C in 5% CO2 and 95% humidity.
The cytotoxicity of the P(MAA-co-CMA) on SH-EP cells was
evaluated using the CellTiter-Blue cell viability assay. The assay was
performed according to the manufacturer’s protocol. In brief, SH-EP
cells were seeded in to 96-well plates at 3 × 103 cells/mL and grown
for 24 h at 37 °C, 5% CO2 in DMEM containing 10% FBS. Cells were
then exposed to varying concentrations of P(MAA-co-CMA) and
incubated for another 72 h. After the incubation period, 20 μL of
CellTiter-Blue was added to each well, and the plate was further
incubated at 37 °C, 5% CO2 for 2−3 h. The fluorescence was recorded
at λex = 540 nm and λem = 590 nm. The effect of the presence of
polymer on the viability of the cells was calculated as follows:
⎧ F − FB ⎫
%cell viability = ⎨ Tr ⎬ × 100
⎩ FUntr − FB ⎭
FTr and FUntr are the fluorescence intensity of treated cells
(treatmented with polymer solutions at varying concentrations) and
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untreated cells (cells only in media, positive control for 100%
viability), respectively. FB, is the fluorescence intensity of blank wells
(wells with media and polymer treatment, no cells) where the values
are similar to 10% Triton X-100 treatment, which corresponds to 0%
viability. Samples were tested in triplicate using cells at different
passage numbers.
■
RESULTS AND DISCUSSION
Copolymerization of tert-Butyl Methacrylate and
Cholesteryl Methacrylate. Owing to the solubility mismatch
between CMA monomer (water insoluble) and methacrylic
acid monomer (water soluble), a two-step reaction route was
adopted for polymerization (Scheme 1, step 1). Cholesterol
was first esterified forming a methacrylate and then used for
copolymerization with t-BMA in the presence of CPABD, a
dithioester RAFT agent, which has been widely used for the
polymerization of methacrylates.56 pH-responsive copolymers
of CMA were then formed by the hydrolysis of t-BMA units of
the copolymers to methacrylic acid units. tert-Butyl ester has
received significant attention as an effective protecting group
due to the labile ester-alkyl bond.57 The tert-butyl protecting
group can be easily removed via acid-catalyzed processes such
as toluene/toluenesulfonic acid,57,58 dioxane/HCl,59 or di-
chloromethane (DCM)/trifluoroacetic acid (TFA) treat-
ments.60,61
GPC traces of P(t-BMA-co-CMA) (obtained at [t-BMA]/
[CMA]/[RAFT]/[AIBN] molar ratio of 200.0/4.0/1.0/0.2,
total monomer concentration of 4.08 M), shown in Figure 1A,
reveals the formation of monomodal molecular weight
distribution during polymerization. The monomodal GPC
chromatograms indicate that the polymerization occurs in the
absence of side reactions, which can cause branched
polydisperse polymers.62 It is evident from GPC traces that
the molecular weight of P(t-BMA-co-CMA) increases with
monomer conversion. The semilogarithmic monomer con-
version (determined by 1H NMR) shown in Figure 1B
indicates that monomer conversion increases concomitantly
with time. The evolution of the Mn with monomer conversion
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Figure 1. Results of RAFT polymerization of P(t-BMA-co-CMA) in toluene at 68 °C ([t-BMA]/[CMA]/[RAFT]/[AIBN] = 200.0/4.0/1.0/0.2).

(A) GPC traces of P(t-BMA-co-CMA) synthesized at different polymerization times (from THF GPC); (B) Ln[M]o/[M] vs time; and (C) number
average molecular weight (Mn)GPC (triangles) and PDI (circles) vs monomer conversion.

(Figure 1C) reveals a linear pseudo-first-order kinetic plot
along with a narrow polydispersity profile of the produced
polymers. As a result, both narrow molecular weight
distributions and linear increase of molecular weights with
monomer conversion indicate that the polymerization of P(t-
BMA-co-CMA) performed at [t-BMA]/[CMA]/[RAFT]/
[AIBN] ratio of 200.0/4.0/1.0/0.2 (4.0M/0.08M/0.02M/4
mM) was a well-controlled polymerization consistent with the
known traits of the RAFT mechanism.
Table 1, which contains polymerization conditions for P(t-
BMA-co-CMA) at varying monomer/RAFT agent ratios, also
confirms that the synthesized polymers hold RAFT-controlled
character. In summary, increasing the monomer/RAFT agent
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molar ratio or the polymerization time at a fixed monomer/
RAFT agent molar ratio result in larger molecular weight
polymers, proving that polymerization of (t-BMA-co-CMA)
could be controlled via the RAFT mechanism.
Purified polymers were analyzed by 1H NMR using CDCl3 as
the solvent (Figure S7A, Supporting Information). Signals at
5.3 and 4.5 ppm, indicative of one olefinic proton in cholesterol
moiety and methine, -COO-CH-, the main link between
cholesterol moiety and ester group, were observed in equal
intensities, suggesting that the cholesterol units stay intact
during polymerization process.
To obtain polymers with varying cholesterol contents,
polymerizations were performed with increasing CMA content
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Biomacromolecules Article

in the feed (Table 2). The composition of the copolymers Conversion of tert-butyl ester to carboxylic acids was
could be calculated from the integrations of relevant proton achieved by treating P(t-BMA-co-CMA) with TFA at room
temperature for 32 h (Scheme 1, step 2). TFA treatment was
Table 2. Copolymers of t-BMA and CMA Prepared at chosen due to its selectivity in cleaving tert-butyl ester groups
Varying Compositions without affecting other types of esters.63 Initially the reaction
was conducted on P(t-BMA) and the specific deprotection of
CMA in the CMA in the
CMA in the copolymer copolymer tert-butyl groups was monitored by 1H NMR at different time
feed (mol (mol % of (wt % of intervals (Scheme S3 and Figures S4 and S5, Supporting
% of total resultant resultant conversionb Mnc Information). Indication of hydrolysis being complete was
monomer)a copolymer) copolymer) (%) (g/mol) PDId
determined by the disappearance of the tert-butyl protons peak
2 2 6 73 16500 1.19
at 1.41 ppm along with the formation of an acidic proton at
4 4 12 76 15800 1.10
12.04 ppm.64 The signals intensity decreased proportional to
8 8 23 69 18000 1.11
time; after 16 h, the tert-butyl group had nearly disappeared,
10 10 28 64 17900 1.14
although the reaction was continued to remove side products
12 12 32 60 18300 1.08
such as CF3C(CH3)3 (1.01 ppm) and OHC(CH3)3 (1.19 ppm,
13 13 34 62 18600 1.27
4.64 ppm). Extended reaction time resulted in a clearer
15 16 40 67 21700 1.23
a
hydrolysis with minimum side products (Figure S4, Supporting
[Total monomer]/[RAFT]/[AIBN] molar ratio in the feed = 204.0/ Information).65 In a control experiment, the effects of acid
1.0/0.2. Total monomer concentration in the feed, 4.08 M; hydrolysis on PCMA were investigated. PCMA was treated
polymerization temperature, 68 °C; solvent, toluene. bMonomer
conversion determined by 1H NMR. cThe number of average with TFA for 32 h (Figure S6, Supporting Information).
molecular weight determined by THF GPC analysis using PS Results indicated that acid hydrolysis conditions applied for the
standards. dPolydispersity index. cleavage of tert-butyl ester had no effect on P(CMA) ensuring
this treatment to be applicable to P(t-BMA-co-CMA).
Subsequently, acid hydrolysis was performed on P(t-BMA-co-
signals in the 1H NMR spectra. The total integration between CMA) and monitored by 1H NMR in DMSO revealing
2.4 and 0.6 ppm illustrates overlapping t-BMA, 14H, and successful deprotection of tert-butyl groups (Figure S7,
cholesterol moiety, 48H peaks. The mole ratio was calculated Supporting Information). The dramatic change in solubility
by the following equation: CMA mol % = [∫ a/(((∫ v − (48 × of the polymers (becoming soluble in aqueous solution) upon
∫ a))/14) + ∫ a)] × 100; t-BMA mol % = [((∫ v − (48 × acid treatment was also an indicator that the ester bonds had
∫ a))/14)/(((∫ v − (48 × ∫ a))/14) + ∫ a)] × 100 (Figure been cleaved. Subsequent to acid hydrolysis, copolymers having
S7A, Supporting Information). According to the NMR analysis 2, 4, and 8 mol % CMA became water-soluble. With the
(Table 2), the copolymer composition (between 60 and 76% increase in cholesterol content (more than 8 mol %), the
monomer conversions) was the same as the comonomer feed, deprotected copolymers, P(MAA-co-CMA), seemed to remain
suggesting a statistical placement of the two monomers along water insoluble. The water-soluble copolymers (having 2, 4,
the copolymer chain and almost equal reactivity of the and 8 mol % CMA) were used for further analysis.
monomers toward both propagating species. In this study, The thermal properties of the copolymers were briefly
statistical copolymers instead of block copolymers were examined via differential scanning calorimetry (DSC). Single
intended to be synthesized to minimize phase segregation of glass transition temperatures (Tg) were observed for homopol-
cholesterol units in aqueous solution (avoid from micellization) ymers and copolymers in the range of 82−228 °C (Figure S8,
and maximize their interaction with lipid membranes. Supporting Information). Poly(cholesteryl methacrylate)

Figure 2. DLS results of the copolymers (0.125 mM) having 2, 4, and 8 mol % CMA in buffer solutions at varying pHs (pH 4.5, 5.5, 6.5, 7.4) at 25
and 37 °C. Anova statistical error values point out the results to be within *P < 0.0005 and **P < 0.005 accuracy.

3069 dx.doi.org/10.1021/bm300846e | Biomacromolecules 2012, 13, 3064−3075

Biomacromolecules Article

(PCMA; Mn,GPC, 4000 g/mol; PDI, 1.13) and poly(methacrylic

acid) (P(MAA; Mn,GPC, 36000 g/mol; PDI, 1.17) displayed Tg
values at 82 and 228 °C respectively, consistent with
literature.50,66 A representative DSC thermogram of copolymer
P(MAA-co-CMA) (8 mol % CMA) exhibited a Tg of 168 °C
valued between the two homopolymer transition temperatures.
The existence of a single glass-transition temperature indicated
the absence of microphase separations, suggesting an overall
statistical copolymer structure.67
The copolymers (at 0.125 mM concentration in isotonic
buffer solutions at varying pH values) were investigated by
dynamic light scattering (DLS) in respect to their composition
and environmental pH (Figure 2). Previously, light scattering
analysis conducted on PMAA demonstrated intrachain
rearrangements, from a coiled conformation to an expanded
Figure 3. Absorbance values at 400 nm of copolymers containing 2
open chain state, between pH 4 and pH 6, triggering the chains mol % CMA (triangles), 4 mol % CMA (circles), and 8 mol % CMA
to swell and increase the polymer size.31,68,69 According to the (diamonds) in differing pH solutions at 0.125 mM concentrations.
results, the number average hydrodynamic diameter of
copolymers having 2 mol % CMA (Mn,GPC, 16500 g/mol; thermore, below the pKa value (∼pH 4) the carboxylic acid
PDI, 1.19) and 4 mol % CMA (Mn,GPC, 15800 g/mol; PDI, groups gradually neutralize, which shift PMAA into a more
1.10) ranges from 5 ± 0.3 to 7 ± 0.4 nm, while for the globular, compact conformation.14,73,76,77 From previous
copolymers having 8 mol % CMA (Mn,GPC, 18000 g/mol; PDI, studies it is clear that the MAA content has a great effect on
1.11) the diameter shifts to the range between 8 ± 1.1 and 13 ± the amphiphilic balance of the copolymers. As the pH of the
1.9 nm at all pH and temperature values investigated. Size solution is increased, the COOH/COO− ratio in the copolymer
distribution plots (Figure S9, Supporting Information) along decreases, which makes the copolymer chains more hydro-
with PDI data (Table S1, Supporting Information) obtained philic.78 In other words, at higher pH, the copolymer chains are
from DLS verify the uniform size of copolymers. in their most soluble state, causing the solution to appear less
The larger hydrodynamic diameter of the copolymer with the turbid. With increasing hydrophobicity (higher CMA content),
highest CMA content suggests the existence of supramolecular the pH-responsive phase transition values progressively rose
organization of the copolymer chains in solution most likely from pH 3.9 in the case of 2 mol % CMA and pH 4.7 in the
due to the hydrophobic interactions (through the CMA units). case of 4 mol % CMA to pH 5.4 in the case of 8 mol % CMA.
It is possible that cholesterol units in the copolymer with 8 mol This result suggests that decrease in PMAA content of
% CMA undergo smectic arrangement leading to the formation polymers increases the pH at which they change their
of bilayered or partially interdigitated packing of two copolymer conformation from a hydrophilic state to a hydrophobic state.20
chains,35 while the copolymers with less CMA content present Atomic Force Microscopy. The larger hydrodynamic
as unimers due to their higher hydrophilicity, as indicated by diameter and the higher turbidity of the copolymer chains with
their relatively smaller hydrodynamic sizes in comparison to 8 the highest CMA content (8 mol %) suggested the self-
mol % CMA. organization of this copolymer in aqueous solutions, as
The hydrodynamic diameter of all copolymers increased with explained above. To investigate this hypothesis, the copolymers
the increase in pH. Results are consistent with literature with 2 mol % and 8 mol % CMA (at 0.125 mM concentration
indicating amphiphilic copolymers with carboxylic acid groups in buffer solution at pH 5.0) were investigated by atomic force
have the propensity to exist in expanded random coil microscopy (AFM) (Figure 4). As expected, the copolymer
conformations at pHs above their pKa, in other words at with 8 mol % CMA content appeared to form spherical
physiologic pH (pH 7.4), the copolymers are in their most supramolecular structures in aqueous solution at pH 5.0. This
extended form due to the charge repulsion between their occurs most likely due to the relatively more hydrophobic
anionic carboxyl groups.70,71 Upon pH change, in acidic character of this copolymer which tends to minimize the
medium below their pKa, polymers tend to protonate forming exposure of the higher number of cholesterol units to solution.
more globular, collapsed structures, compared to their original
conformation at neutral pH.70,72−74 This transition can be
observed by the decrease in the hydrodynamic radius of the
polymer chains, as seen in Figure 2.75 The temperature can also
affect the hydrodynamic diameter of the copolymers. The
temperature change from room temperature to 37 °C (body
temperature) at constant pH influences the polymer chains to
expand, increasing their diameter.
Turbidity Assay. The pH-responsive phase behavior of
P(MAA-co-CMA), comprised with varying CMA units, was
determined using UV−vis spectroscopy. The change in
absorbance values at 400 nm for the copolymers in different
pH solutions at 0.125 mM concentration is shown in Figure 3. Figure 4. Atomic force microscopy images of the copolymers with (A)
PMAA, bearing carboxylic acid groups, when ionized at pH 6 2 mol % CMA and (B) 8 mol % CMA. The sample solutions (0.125
(above pKa) exists in an expanded form that is dominated by mM) in buffer at pH 5.0 were dropped on silicone substrate and dried
Coulomb repulsions between the carboxylate anions. Fur- at room conditions.

3070 dx.doi.org/10.1021/bm300846e | Biomacromolecules 2012, 13, 3064−3075

Biomacromolecules Article

Figure 5. Surface plasmon resonance (SPR) real-time monitoring of binding of the copolymers with 2, 4, or 8 mol % CMA onto cell plasma
membrane-mimicking lipid bilayer (cholesterol/phosphatidylcholine molar ratio of 0.42). Polymers (0.5 mM) in buffer solutions at pH 5.0 or 7.4
were injected over the lipid bilayer surface at a flow rate of 20 μL/min for 6.5 min. After reaching saturation, surfaces were rinsed (shown by arrow)
with related buffer solutions for the same period of time.

Figure 6. SPR real-time monitoring the binding of the 2 mol % CMA copolymer onto lipid bilayer mimicking the plasma (PM) or endosome
membrane (EM) in acidic or neutral conditions. PM consisted of cholesterol/phosphatidylcholine constituents at a cholesterol/phosphatidylcholine
molar ratio of 0.42, and EM consisted of cholesterol and phosphatidylcholine at a mole ratio of 0.80. The related polymer solution (0.5 mM) was
injected over the lipid bilayer surface at a flow rate of 20 μL/min for 6.5 min; upon reaching saturation, it was then rinsed with related buffer solution
(indicated by the arrow).

In contrast, the copolymer with 2 mol % CMA appeared to
exist in extended chain conformation because of its higher
hydrophilicity compared to the copolymer having 8 mol %
CMA. This result was in good accord with the results obtained
from DLS and turbidity experiments.
Surface Plasmon Resonance. The interactions between
polymer molecules and lipid bilayer immobilized surfaces were
investigated by SPR. Lipid bilayer comprised of varying
cholesterol/phosphatidylcholine constituents were generated
as cellular membrane models for the plasma and endosome
membranes.54 Polymers at 0.5 mM concentration, composed of
varying cholesterol units (2, 4, and 8 mol % CMA), were
interacted first with the plasma membrane-mimicking surfaces
in slightly acidic and neutral pH. Surfaces were treated with
polymer solutions of 0.5 mM concentration to ensure
interaction of the polymer with lipid layer and obtain complete
3071
surface coverage.79 Figure 5 displays the interaction of the
copolymer containing 2 mol % CMA with lipid bilayer surface
at both pHs to be significantly higher than the interactions of 4
and 8 mol % CMA containing copolymers. As supported by
AFM analysis, the higher hydrophobicity of the copolymers
with 4 and 8 mol % CMA compared to 2 mol % CMA
copolymer might be causing the self-organization of the
copolymer chains in aqueous solution to minimize the exposure
of hydrophobic cholesterol units to water, which in total
reduces the interactions of the cholesterol units along the
chains with the lipid layer and, thus, demonstrating lower
binding profile. Furthermore, in acidic medium, the copolymer
having 2 mol % CMA exhibits almost five times greater binding
when compared with the binding signal intensity of the same
copolymer at neutral pH. The increase in surface binding at
lower pH can be explained by the relatively higher hydro-
dx.doi.org/10.1021/bm300846e | Biomacromolecules 2012, 13, 3064−3075
Biomacromolecules Article

phobicity (exposed cholesterol moieties) along with the lower membrane-lytic effect of the copolymer containing 2 mol %
ionic charge (protonated MAA units) of the copolymer, CMA, was found to be the strongest (Figure 7). This result, in
possibly increasing the interaction of the copolymer chains
with phosphatidylcholine/cholesterol lipid bilayer.74,80
Upon determining the copolymer composition displaying the
highest binding to the plasma membrane-mimicking surface,
the polymer−lipid interaction was further investigated in
relation to lipid bilayer composition (Figure 6). The interaction
of the 2 mol % CMA copolymer with plasma or endosome
membrane-mimicking lipid bilayer arrangements having differ-
ent cholesterol contents (molar ratio of 0.42 and 0.80,
respectively) were compared in a pH-dependent manner
using SPR. The binding of the copolymer with both lipid
bilayers at acidic pH was higher when compared with the
binding at neutral pH, as expected. Interestingly, the binding at
acidic condition with the plasma membrane-mimicking bilayer
was higher than the binding with endosome membrane-
mimicking bilayer which had almost 2-fold higher cholesterol
content. This was attributed to the possible interactions formed
among protonated MAA units of the copolymer and
phophatidylcholine components in the lipid bilayer surfaces.
Intermolecular hydrogen bonding created at acidic pH between
phosphatidylcholine units (higher content in plasma mem-
brane-mimicking bilayer) and copolymer chains is likely to
dominate intra- and intermolecular hydrogen bonds between
MAA units along copolymer chains, consequently leading to
greater interactions with the lipid bilayer.81,82 It is also possible
that the higher rigidity of the endosome membrane-mimicking
bilayer because of the higher cholesterol content decreases the
interaction of the bilayer with the polymer chains. Consistent
with these results, at pH 7.4 there was almost no binding with Figure 7. Results of liposome lysis assay. The lysis obtained with the
both membrane models, possibly because of the electrostatic copolymers containing 2, 4, and 8 mol % CMA are shown in the
repulsion between negatively charged copolymers and anionic graph, PMAA represents lysis gained by poly(methacrylic acid) only
lipid bilayer. (with no cholesterol units), and 10% Triton X-100 (T) signifies
Liposome Lysis Assay. The extent of pH-dependent maximum lysis. Polymer concentrations are (A) 1.5 or (B) 3.0 μM.
hydrophobic association of the copolymers has been identified The results are the average of two different sets of experiments
performed in triplicate. Error bars represent standard deviation. Anova
as an important factor in determining the degree of membrane statistical analysis results reveal the values to be within *P < 0.05 and
destabilization activity which is required for intracellular drug **P < 0.006 accuracy.
delivery vehicles.83 Amphiphilic copolymers containing 2, 4,
and 8 mol % CMA, were analyzed for their pH-dependent
membrane-lytic properties using a liposome leakage assay, as accord with the SPR data reveals that the copolymer having 2
described in the literature.84 The assay was first performed by mol % CMA exhibits the greatest polymer−lipid interactions
treating the SRB-loaded liposomes with copolymer solutions at and displays the highest membrane destabilization activity at
0.125 mM concentrations at pH 5.0 and 7.4. Complete release pH 5.0. The lower membrane-lytic activity of the copolymers
of SRB from liposomes was observed with no pH-dependent with higher CMA content can be attributed to the self-
profile (data not shown). This suggests that the copolymers at organization of these copolymers in solution, minimizing the
0.125 mM cause nonspecific (pH-independent) destabilization hydrophobic interactions between copolymer CMA units and
of liposomes causing the leakage of SRB at both pH values liposomes. Amphiphilic polymers have been shown to interact
tested (pH 5.0 and 7.4). Here it should be noted that the with lipid vesicles by inserting their hydrophobic segments into
copolymers at a higher concentration (0.5 mM) show pH- the lipid layer as a result of the hydrophobic interactions.85
dependent interaction with the lipid layer anchored onto a The liposome lysis by the copolymers was observed to be
surface (as SPR results indicated). The discrepancy between pH-dependent.86,87 The membrane-lytic activity at pH 5.0 was
the results obtained from SPR and liposome leakage assay 3-fold (in the case of 2 mol % CMA), 2-fold (4 mol % CMA),
might be due to the fact that the lipid assembly on the surface is and 1.4-fold higher (8 mol % CMA) than the lysis at pH 7.4.
well-packed and, thus, less flexible than the lipid layer of The copolymers displayed an average of 34% SRB leakage at
liposomes, which minimizes the interactions with the neutral pH. Similar residual lytic activities at neutral pH have
copolymer chains at ionized (hydrophilic) state (at pH 7.4) also been reported previously for various pH-dependent
even at high concentrations such as 0.5 mM. membrane disruptive polymers.21,70,88 As the pH of the
The liposome leakage assay was repeated using copolymer solution is increased, the copolymer chains become negatively
solutions at much lower concentrations (1.5 and 3.0 μM) to charged and more hydrophilic because of the ionization of the
avoid from nonspecific (pH-independent) destabilization of carboxylic acid groups of MAA units.78 This decreases the
liposomes. Based on the release profiles of SRB from liposomes interactions between phophatidylcholine liposomes and the
after incubation with the copolymers, the pH-dependent copolymer chains. Hydrogen bonds that may form at acidic
3072 dx.doi.org/10.1021/bm300846e | Biomacromolecules 2012, 13, 3064−3075
Biomacromolecules
pHs between the carboxylate groups of the copolymer chains
and the phosphate groups of phophatidylcholine molecules
may also help the destabilization of the membrane leading to
SRB leakage from the liposome. The deficiency of lysis activity
in our control group, PMAA acidic polymer, shows that the
membrane interaction is mediated by the copolymers hydro-
phobic cholesterol units. In summary, both hydrophobic
interactions and H-bonding play a role in lipid membrane
destabilization; however, the significant ratio of these two
interactions present in a polymer dictates the aptitude of
membrane disruption. Changing the polymer concentration
from 1.5 to 3.0 μM did not have an effect on the extent of lysis
(Figure 7). Overall results suggest that the copolymers of MAA
and CMA are membrane-lytic, and their lysis ability differs
according to their CMA content and pH of the environment.
Moreover, the pH-dependent lysis ability of the copolymer with
2 mol % CMA potentially demonstrates the desired profile of
endosomolytic polymers used for intracellular drug deliv-
ery.16,20,26,71
Determination of Cell Viability via CellTiter-Blue
Assay. Finally, P(MAA-co-CMA), with varying CMA contents,
were characterized with regard to their effects on viability of
human neuroblastoma SH-EP cells by CellTiter-Blue assay.
This assay is based on the ability of viable cells to convert the
redox dye resazurin into the fluorescent end product, resorufin.
Results of in vitro viability experiments after 72 h of incubation
of the cells with different concentrations of polymers, ranging
from 0.06 to 30 μM (per 3 × 103 cells), are shown in Figure 8.
Cell viability (%) is expressed as a function against the
untreated cells (control for 100% viability) and 10% Triton X-
100 treatment (control for 0% viability).
Figure 8. Viability of human neuroblastoma SH-EP cells after
incubation with P(MAA-co-CMA) copolymers that consist of 2, 4,
and 8 mol % CMA along with P(MAA) for 72 h, measured with
CellTiter-Blue assay. The assay was repeated three times in triplicate
and the viability results were normalized according to the positive
control (untreated cells). Error bars represent standard deviation.
Anova statistical error values point out the results to be within *P <
0.06 and **P < 0.001 accuracy.
Statistical copolymers composed of 2, 4, and 8 mol % CMA
along with P(MAA) at the concentrations tested displayed
similar behavior on the cells. The viability of the cells for 30 μM
to 0.06 μM treatments varied between 86 and 100%, indicating
no significant toxicity of the copolymers.
3073
■
Article
CONCLUSION
In summary, well-defined statistical copolymers of P(MAA-co-
CMA), with varying cholesterol content, were synthesized via
RAFT polymerization. Controlled composition and narrow
molecular weight distributions were obtained for both
homopolymers and statistical copolymers. The shift in turbidity
of copolymer solutions together with the change in hydro-
dynamic radii up on pH adjustments revealed the pH-
responsive phase behavior of the copolymers. SPR studies
along with the liposome leakage assay indicated that these
copolymers, depending on their CMA content and the pH, can
interact with the lipid bilayer. In vitro cell viability assays,
conducted on human neuroblastoma SH-EP cell line, evidenced
that these copolymers display no substantial toxicity at the
concentrations tested.
These results support the value of P(MAA-co-CMA) for
further investigating their use in the development of intra-
cellular drug delivery vehicles. It would be possible to
incorporate cationic substitutes with these new anionic
polymers and negatively charged RNA/DNA based therapeu-
tics. Future work will focus on the investigation of the
copolymer molecular weight on the membrane-lytic activity and
the preparation of P(MAA-co-CMA) containing polyplex
formulations with difficult-to-deliver biological macromolecules,
such as short interfering RNA (siRNA) and DNA, and the
testing of their efficacy in cytoplasmic delivery.
■
ASSOCIATED CONTENT
* Supporting Information
S
Experimental methods for synthesis of cholesteryl methacrylate
(CMA) monomer, homopolymerizations of CMA and t-BMA,
and acid hydrolysis, differential scanning calorimetry traces, and
representative size distribution plots of copolymers, Schemes
S1−S3, Figures S1−S9, and Table S1. This material is available
free of charge via the Internet at http://pubs.acs.org.
■ AUTHOR INFORMATION
Corresponding Author
*Tel.: +90-232-750 6660. E-mail: volgabulmus@iyte.edu.tr.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
The authors acknowledge The University of New South Wales
Gold Star Award and Dr. Ling-Jiun Wong for her help on
preliminary literature search. V.B. also acknowledges The
Scientific and Technological Research Council of Turkey
(TUBITAK) for research Grant No. 111T960.
■
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